1. Field of the Invention
The present invention is directed to a system which dramatically increases the speed and efficiency by which substances can be tested for their effects upon a myriad of biochemical processes, for example in living cells. The system can be applied to many fields including application in high throughput drug screening. When applied to the field of high throughput drug screening the system only requires a fraction of the cells currently needed for such tests, enables microminiturization of the process, and reduces the cost of drug screening by reducing the amount of reagents, cells, and disposable materials utilized in the screening process.
2. Discussion of the Background
Scientific research in general, and medical research as a specific example, often requires the evaluation of certain compositions relative to other compositions, plant cells, animal cells, etc. A common example of such research would be in the discovery and development of new drugs.
The discovery and development of a new drug occurs via two main stages. An initial discovery stage aims to the identification and optimization of chemical lead structures among the numerous compounds synthesized to interact with a molecular target putatively involved in the pathophysiology of a human disease. A development stage then follows that assesses the pharmacokinetics, safety and efficacy properties of those drugs found to be potential candidate in humans. Recent advances in drug discovery include the synergistic development of two new technologies in biomedical research known as Combinatorial Chemistry (CC) and High Throughput Screening (HTS). CC, via computer-aided drug design and automated organic synthesis, allows thousands of compounds (a library) of systematic variants of a parent chemical structure to be produced in parallel. Pharmaceutical researchers can now create in a relatively short time millions of new compounds designed to target a specific cellular substrate such as receptors, enzymes, structural proteins and DNA, thus increasing the need for rapid and broadly applicable methods to screen these compounds. While it is important to screen compounds for the targets they were designed for, it is also important to be able to screen compounds for their unintended targets to anticipate potential side effects of selected candidate drugs and to find new uses for these substances if the side effect turns out to be a desired property. The development of HTS has been making it feasible, through automation and miniaturization techniques, to screen upwards to millions of drug candidates a year with robotic workstations running continuously 24 hours a day, 7 days a week. Billions of animal cells expressing the molecular target against which a library is made are grown in 96, 384, or 1536 micro-well plates and, via automated drug and liquid delivery and computerized read-out devices, are tested for a biological response to the drugs.
In conventional HTS systems, animal cells are placed in each of the individual wells of the micro-well plates and are subject to many different processes to test for a response to applied drug candidates. However, an extremely large number of novel drug candidates can now be made available by CC. The conventional approach in HTS systems has been to increase the number of individual wells in the micro-well plates to increase the number of drug candidates that can be screened at one time.
The Scintillation Proximity Assay by Amersham, as disclosed in U.S. Pat. No. 4,271,139 and U.S. Pat. No. 4,382,074 as examples, is a one-step radioisotope-based assay that can be easily automated for HTS. However, the advantages of this sensitive and simple technique are challenged by increasing constrains on the use as well as the cost of disposal of radioactive materials. Thus, new nonradioisotope based screening alternatives have been sought. The development of fluorescent probes able to penetrate living cells, or be biochemically synthesized by cells, such as with chimeric constructs of green fluorescent proteins (GFP), and target protein receptors and enzymes in combination with improved optical instrumentation and means of delivering light and detecting signals has made fluorescence based technique the preferred alternative for many research applications. Fluorimetric Imaging Plate Reader (FLIPR) is a recently developed technique which permits kinetic measurements of intracellular fluorescence on cells labeled with an indicator whose fluorescence properties change upon binding to a cellular substrate targeted by a given drug. FLIPR allows for simultaneous and real time measurements of 96 (and recently 384) samples every second and finds an ideal application in HTS for candidate drugs targeting cell membrane receptors or channels whose activation leads to intracellular ion fluxes in a matter of seconds as in the case of the internal release or influx of calcium ions. In the pharmaceutical industry, HTS is currently performed on commercially available cell lines established from a variety of embryonic and adult animal tissues both normal and pathological. To create cell lines, cells are made immortal via exposure to defined agents such as viruses or chemicals thus acquiring the ability to continuously grow and divide in culture. However, it is generally recognized that, as a result of the immortalization procedure, changes in the expression of certain genes can randomly occur leading to a cell phenotype which might deviate from that of the parental tissue. For example, immortalized liver cells might have lost the ability to express a certain receptor, or to express it in the correct form or cellular compartment as the parental liver cells. Consequently, upon establishment, cell lines are tested for the expression of specific markers, receptors, enzymes, etc. and categorized accordingly.
In contrast to immortal cell lines, primary cell cultures derive from cells freshly isolated from a given organ or tissue. No viral or chemical intervention are used to pressure the cell division cycle and, thus, the cells will survive in vitro for only a short period of time, generally 10-15 days, and need to be re-established quite frequently during a research project. Primary cells are obtainable from a variety of animal models as well as human tissues surgically removed mainly for pathological reasons. Because of their short life span, primary cells maintain the biological stigmata of the original tissue virtually unchanged and, thus, are the research model considered closest to the in vivo environment. Therefore, drug screening on primary cells is highly desirable because it both decreases the chances to miss a valuable lead and increases the physiological relevance of the data collected. However, the dependence of conventional HTS on a tremendously high volume of biological substratexe2x80x94billions of cells grown and processed in 96-, 384-, or 1536-micro-well platesxe2x80x94has prevented the application of widespread drug screening to primary cells because they are only available in limited quantities. Thus, cell lines exhibiting the biological target against which a drug library has been made are the unique and invaluable source of biological substrate fitting the needs of HTS currently available in drug discovery.
In many of the currently available HTS methodologiesxe2x80x94e.g. fluorescence imaging basedxe2x80x94the vast majority of cells grown are wasted because, among all the cells present in a given well and exposed to a drug candidate, only those occupying a microscopic field are ultimately monitored for their response. Along with the cells, precious chemical compounds and expensive reagents and supplies are dissipated making the process wasteful and time-consuming, thus reducing the overall afforded by HTS. As discussed above, conventional HTS systems provide individual cells in individual wells of micro-well plates. FIG. 1(a) shows a standard 96-well micro-well plate 100 including 96 individual wells 110, and an individual well 110 is shown in FIG. 1(b). Each micro-well 110 has a diameter D, which in the example of the standard 96 well plate 100 is 6 mm.
Currently available HTS systems perform the screening on the micro-well plates with a process such as shown for example in FIG. 2. In an example of utilizing the 96 well format in a first step S20, as shown in FIG. 2, cells are plated under aseptic sterile conditions and grown into each of the 96 wells. During the growth phase under aseptic sterile conditions, removal of growth media must be made from each well and new media repipetted into each well under aseptic sterile conditions. Once the cells are grown, then the wells are treated in step S25, which may include, as an example, loading the cells with a fluorescent dye, which again requires removal of media from each well, addition of the dye, incubation for a period of time, etc. Then, in step S30 rinsing of the cells is executed for, as an example, removal of dye from each well. Finally drug candidates are added to each well and the cell response is measured in step S35 and the plates are then discarded in step S40.
The conventional HTS process shown in FIG. 2 suffers from the following drawbacks. First, in that process cells are grown into the entire area of each of the 96 wells, which means that that entire area of each of the 96 wells must be loaded with the fluorescent dye, the drug candidate, and any other reagents needed. Further, in the conventional HTS process of FIG. 2 as there are only 96 wells only 96 drug candidates can be evaluated at a single time. Although that may be a significant number of drug candidates, the HTS system relies on evaluating tens of thousands of drug candidates to determine whether the drug candidates provide a desired reaction with the cells. Therefore, evaluating only 96 drug candidates at one time is very time consuming evaluation process.
Accordingly, one object of the present invention is to provide a novel analysis system for analyzing a sample in a highly efficient manner.
A more specific object of the present invention is to provide a novel cost effective HTS system which greatly improves the efficiency, throughput, and physiological relevance of HTS drug screening.
A further object of the present invention is to provide a novel HTS system which dramatically reduces the number of cells used for each measurement and which also reduces the amount of reagents and disposable materials used in the HTS process.
A further object of the present invention is to provide a novel HTS system which can be effectively used with primary cells in addition to immortal cells.
The mainstream of the pharmaceutical industry is moving to solve HTS throughput problems by developing multiwell plates with more, and thus smaller, individual wells per plate. The current trend in the HTS industry is to move from 96 well plates 100 such as shown in FIG. 1 to 1536 well plates, a 16 fold increase in the number of wells per plate and a 16 fold decrease in the size of each individual well. Coincident with this is increased complexity: 1) of growing cells in the smaller wells, 2) in optics, 3) in fluid handling, and 4) of the mechanics involved with the process-all under aseptic sterile conditions. These drawbacks are in addition to the expenditure of untold hundreds of millions of dollars to achieve probably less than an order of magnitude increase in speed without other significant technological advantages which would increase the information content of the screening process.
The inventor of the present invention, however, has taken a contrary approach to that taken by the mainstream in the pharmaceutical industry. The inventor of the present invention has specifically not taken an approach to reduce the size of a micro-well, but has taken an opposite approach which can maintain the existing well structures, and in fact with the novel HTS system of the present invention cells can even be grown on monolayers without any predetermined well structure.
To achieve the above and other objects, the novel HTS system of the present invention tests the action of a drug candidate upon a group of cells in a monolayer such that a microscopic field area of 100-200 microns in diameter is isolated from other cells on the monolayer by creating a seal between a drug delivery perfusion unit and the cells to create a microspace for analysis. The novel HTS system of the present invention can provide improved efficiency over current HTS methods since the vast majority of the cells on a monolayer can be used for drug testing rather than wasting most of the cells and reagents, as is currently the case with HTS technology based upon cells grown in multi-well plates.
Further, the novel HTS system of the present invention can provide improved efficiency over current HTS systems since the HTS system of the present invention can more readily be used with primary cells as a first screen rather than requiring immortal cells for such an initial screening of compounds. Primary cells have exactly the same biological characteristics as do any cell in the body, because in fact that is exactly what they are, cells isolated from an animal and kept in cell culture for a short time. Because the number of primary cells available is somewhat limited, the efficient use of cells by the present invention makes it feasible to use primary cells for HTS with the present invention.